Instant infusion pasteurisation of bovine milk. II. Effects on indigenous milk enzymes activity and whey protein denaturation

Direct heat treatment of two milk types, skimmed and nonstandardised full‐fat, was performed by instant steam infusion and compared with indirect heating. Infusion conditions were temperatures of 72–120°C combined with holding times of 100–700 ms, and indirect heat conditions were 72°C/15 s and 85°C/30 s. The activity of indigenous enzymes such as alkaline phosphatase, lactoperoxidase, xanthine oxidase and γ‐glutamyl transpeptidase was evaluated. Infusion temperature was the main determinant of inactivation. Whey protein denaturation represented by β‐lactoglobulin increased significantly with infusion temperature. The nonstandardised milk had a higher denaturation rate than skimmed milk. The effect of instant infusion on pH and milk fat globule size in relation to whey protein denaturation and association is discussed.     

Effect of high-pressure treatment on denaturation of bovine lactoferrin and lactoperoxidase

Lactoferrin and lactoperoxidase are whey proteins with biological properties that may provide health benefits to consumers. These properties are vulnerable to potentially denaturing conditions during processing. High-pressure treatment is an appealing alternative to the traditional heat processing of foods because it exerts an antimicrobial effect without changing the sensory and nutritional quality of foods. In this work, the effect of high-pressure treatment on the denaturation of lactoferrin and lactoperoxidase present in skim milk and whey, and as isolated proteins in buffer, was studied over a pressure range of 450 to 700 MPa at 20°C. Denaturation of lactoferrin was measured by the loss of reactivity with their specific antibodies using a sandwich ELISA. Denaturation of lactoperoxidase was determined by measuring the loss of enzymatic activity using a spectrophotometric technique. No substantial inactivation of lactoperoxidase was observed in any treatment assayed. The concentration of the residual immunoreactive lactoferrin after each pressure treatment was determined, and the data were subjected to kinetic analysis to obtain D and Z values. Denaturation of lactoferrin increased with pressure and holding time, and D values were lower when lactoferrin was treated in whey than in milk, and lower in both whey and milk than in phosphate buffer. Thus, protein is denatured more slowly in buffer and in milk than in whey. Denaturation of lactoferrin in the 3 media was found to follow a reaction order of n=1.5. Volumes of activation of about -34.77, -24.35, and -24.09 mL/mol were obtained for lactoferrin treated in skim milk, whey, and buffer, respectively, indicating a decrease in protein volume under pressure.     

A kinetic model for whey protein denaturation at different moisture contents and temperatures

The denaturation of whey protein samples that had previously undergone heat-treatment for different times at different temperatures and moisture contents was analysed by differential scanning calorimetry (DSC), using the DSC enthalpy as a measure of residual undenatured protein. Data were fitted to first order irreversible or reversible kinetic expressions, and the resulting rate constants were found to increase with both temperature and moisture content. The whole data set was then fitted as a function of time, temperature and moisture content, with rate constants varying according to either Arrhenius or Williams-Landel-Ferry (WLF) kinetics and with selected fit parameters made empirical functions of moisture content. The best fits were obtained using reversible WLF kinetics, which could be further slightly simplified without loss of accuracy. The model provides a platform for single- and multi-objective drying trajectory optimisation with respect to protein denaturation in dairy products.     

Astringency of bovine milk whey protein

Whey protein solutions at pH 3.5 elicited an astringent taste sensation. The astringency of whey protein isolate (WPI), the process whey protein (PWP) that was prepared by heating WPI at pH 7.0, and the process whey protein prepared at pH 3.5 (aPWP) were adjusted to pH 3.5 and evaluated by 2 sensory analyses (the threshold method and the scalar scoring method) and an instrumental analysis (taste sensor method). The taste-stimulating effects of bovine and porcine gelatin were also evaluated. The threshold value of astringency of WPI, PWP, and aPWP was 1.5, 1.0, and 0.7 mg/mL, respectively, whereas the gelatins did not give definite astringency. It was confirmed by the scalar scoring method that the astringency of these proteins increased with the increase in protein concentration, and these proteins elicited strong astringency at 10 mg/mL under acidic conditions. On the other hand, the astringency was not elicited at pH 3.5 by 2 types of gelatin. A taste sensor gave specific values for whey proteins at pH 3.5, which corresponded well to those obtained by the sensory analysis. Elicitation of astringency induced by whey protein under acidic conditions would be caused by aggregation and precipitation of protein molecules in the mouth.     

Effect of mastitis on proteolytic activity in bovine milk

Proteolytic activity of milk was studied before, during, and after experimental-induced mastitis. An inoculum of Streptococcus agalactiae was infused into one quarter of each udder of six cows to elicit an infection. Bacteriological cultures and SCC of milk were used to monitor infection status. Sodium dodecyl sulfate-PAGE was used to measure proteolytic activity of milk. Inhibitor 6-amino-n-hexanoic acid was used to determine the relative proportion of plasmin and nonplasmin proteolytic activity of milk. Somatic cell count, total milk proteolytic activity, and nonplasmin proteolytic activity were higher in infected quarters than in quarters preinfection. After elimination of infections, SCC and nonplasmin proteolytic activity decreased to preinfection amounts. Total proteolytic activity of milk decreased after infections were cured but remained significantly higher than preinfection activity. This postinfection proteolytic activity in milk may be due to an increase in milk plasmin activity. Our data suggest that detrimental effects of mastitis on milk quality can continue after infection has been eliminated and milk SCC have returned to low values.     

The thermal denaturation of the total whey protein in reconstituted whole milk

The kinetic parameters for thermal denaturation of the total whey proteins in whole milk were determined. Denaturation was a second‐order reaction, and an Arrhenius plot showed a change in slope at ~85 °C. At 70–85 °C, the activation energy, enthalpy and entropy were in the range expected for denaturation processes, whereas at 85–115 °C, these parameters were typical for chemical reactions such as aggregation. Equations to predict the denaturation after heating were developed and tested on a range of independently prepared milk samples. There was a good agreement between the predicted and the experimentally determined denaturation levels.     

Yak milk whey protein denaturation and casein micelle disaggregation/aggregation at different pH and temperature

At the natural pH of yak milk (pH 6.6), a low level (<30%) of κ-casein (κ-CN) was found in the serum phase after heating at 95 °C for 30 min, indicating that as much as 70% of the β-lactoglobulin (β-Lg) and κ-CN complexes is associated with the micelle colloidal particles. The β-Lg and κ-CN levels increased from 13.2% and 2.6% at pH 6.0 to 35.2% and 60.1% at pH 7.0, respectively, when yak milk was heated at 95 °C for 30 min. At pH 6.0–6.4, the denatured whey proteins were associated with the caseins in the colloidal phase, resulting in milk gelation upon heating. The distribution of β-Lg and κ-CN complexes increased in the serum phase, demonstrated by the increasing levels of both β-Lg and κ-CN with increasing pH; at high pH (6.6–7.0), large proportions of β-Lg and α-lactalbumin were lost, presumably forming complexes in the colloidal phase.     

Effect of bovine skim milk and whey on monocyte function

Previous studies have documented the ability of bovine milk to inhibit lymphocyte proliferation in response to mitogens. It is not known whether inhibition of lymphocyte proliferation is mediated through the action of monocytes. To address this question, we examined the ability of bovine skim milk and whey to affect monocyte function with emphasis on expression of major histocompatibility class II antigens and production of interleukin-1 by monocytes. Data showed that expression of major histocompatibility complex class II molecules and production of interleukin-1 by monocytes were not altered when monocytes were cultured in the presence of bovine skim milk or whey. Thus, it is unlikely that the suppressive effect of milk on lymphocyte proliferation could be mediated through alterations in the expression of major histocompatibility class II molecules or in production of interleukin-1 by monocytes. The role of other monocyte antigens or secretory products, however, should also be evaluated.     

High pressure-induced denaturation of alpha-lactalbumin and beta-lactoglobulin in bovine milk and whey: a possible mechanism

In this study, high pressure (HP)-induced denaturation of alpha-lactalbumin (alpha-la) and beta-lactoglobulin (beta-lg) in dairy systems was examined. In both milk and whey, beta-lg was less baroresistant than alpha-la; both proteins were considerably more resistant to HP-induced denaturation in whey than in milk. HP-induced denaturation of alpha-la and beta-lg increased with increasing proportion of milk in mixtures of milk and whey. Addition of a sulphydryl-oxidising agent, KlO3, to milk or whey increased HP-induced denaturation of beta-lg, but reduced the denaturation of alpha-la. Denaturation of both alpha-la and beta-lg was prevented by adding a sulphydryl-blocking agent, N-ethylmaleimide, to milk or whey prior to HP treatment, highlighting the crucial role of sulphydryl-disulphide interchange reactions in HP-induced denaturation of alpha-la and beta-lg. Removal of colloidal calcium phosphate from milk also reduced HP-induced denaturation of alpha-la and beta-lg significantly. The higher level of HP-induced denaturation of alpha-la and beta-lg in milk than in whey may be the result of the abscence of the casein micelles and colloidal calcium phosphate from whey, which facilitate HP-induced denaturation of alpha-la and beta-lg in milk.     

The effect of protein profile and preheating on denaturation of whey proteins and development of viscosity in milk protein beverages during heat treatment

The effect of preheat temperature (63 or 77 °C for 30 s; final heat 120 °C for 30 s) and casein to whey protein ratio on the physical characteristics of 3.3%, w/w, dairy protein beverages was investigated. Dispersions preheated at 77 °C had lower viscosity than dispersions preheated at 63 °C. Casein‐containing dispersions had significantly lower levels of α‐lactalbumin denaturation than whey protein‐only dispersions. A higher proportion of casein improved the thermal stability of protein dispersions. Overall, alteration of preheat temperature and casein to whey protein ratio can influence dairy beverage quality, with increasing levels of casein reducing physical changes due to heat treatment.     

Effects of high pressure treatment on indigenous enzymes in bovine milk: Reaction kinetics,inactivation and potential application

High pressure-induced inactivation of the indigenous milk enzymes alkaline phosphatase (ALP), γ-glutamyltransferase (GGT) and phosphohexoseisomerase (PHI) was studied in the pressure range 400–800 MPa at temperatures between 5 and 40 °C. With respect to pressure stability the following ranking was observed: ALP>GGT>PHI. PHI was inactivated after pressure treatment at 500 MPa and 20 °C for 10 min. In terms of reaction kinetics, inactivation of GGT followed first-order reaction kinetics in the range of 400–800 MPa whereas a reaction order of 1.5 was found for ALP. Reactivation of pressure-treated ALP was observed at low enzyme activity resulting from severe pressure treatment and 2 h storage at 35 °C. The influence of process temperature on the pressure-induced inactivation of GGT and ALP was limited in the range 5–40 °C.     

Thermal denaturation kinetics of whey proteins at high protein concentrations

A detailed kinetic study of the thermal reaction kinetics of whey protein concentrate was conducted at high protein concentrations. Whey protein solutions with protein concentrations of up to 40% (w/w) were heated at different temperatures for varying periods of times. The denaturation of β-lactoglobulin followed a reaction order of 1.5 and depended strongly on temperature and protein concentration. The rate of denaturation was shown to increase with increasing temperature. This could be explained by the strong influence of the temperature on the unfolding reaction. Furthermore, the protein concentration induced a faster thermal denaturation, most likely due to the increased probability of collision between whey protein molecules with increasing protein concentration which promotes protein aggregation. The results of this study are of industrial relevance for extrusion processes and the production of protein concentrates in evaporators where high protein concentrations are frequently used.     

Production of low antigenic cheese whey protein hydrolysates using mixed proteolytic enzymes

This study examined the effect of different proteolytic enzymes on the production of cheese whey protein (CWP) hydrolysates with low antigenicity. Four enzyme combinations (1:1) trypsin + papain W‐40 (TP), trypsin + neutrase 1.5 (TN), papain W‐40 + protease S (PP) and papain W‐40 + neutrase 1.5 (PN) were added at the rate of 1% of the CWP and it was incubated for 15, 30, 60, 90, 120 and 180 min at 50 °C. CWP hydrolysis and its non‐protein nitrogen concentrations were higher with TP and TN compared with PP and PN at all incubation times. The SDS‐PAGE revealed complete removal of α‐lactalbumin (α‐LA) and β‐lactoglobulin (β‐LG) from hydrolysates produced by trypsin‐containing enzyme mixtures. Reverse‐phase HPLC analysis ascertained the CWP hydrolysis and SDS‐PAGE results. The lowest antigenicity in CWP hydrolysates was observed with the use of trypsin‐containing enzyme mixtures compared with other enzyme combinations. Present results suggested that TP and TN combinations were the most effective for CWP hydrolysis for the removal of β‐LG from CWP. Further research is warranted to identify the peptides in CWP hydrolysates produced with these enzyme combinations that may help enhance the utilisation of whey protein in human food. Copyright © 2007 Society of Chemical Industry     

变性对转谷氨酰胺酶在乳蛋白间交联影响初探

利用SDS-PAGE电泳结合凝胶成像分析技术，比较了在非变性、加入还原剂变性和加热后再加入还原剂变性三种条件下转谷氨酰胺酶对酪蛋白和乳清蛋白之间的交联情况。结果表明：在非变性条件下，酪蛋白质量分数下降96％，乳清蛋白下降15％，酪蛋白和乳清蛋白几乎不能交联。超分子量聚合物是酪蛋白单一聚合物，α-乳白蛋白形成部分低聚体；在加入还原剂时，酪蛋白质量分数下降86％，乳清蛋白下降30％，反应4h后有少量乳清蛋白和酪蛋白中某一组分交联；预热更有助于酪蛋白和乳清蛋白聚合，在第三种条件下，反应24h后乳清蛋白下降60％。     

Effect of frozen storage on protein denaturation in bovine muscle.

The effect of frozen storage on the solubility, viscosity, rheological and electrophoretic behaviour of myofibrils was studied. The solubility, viscosity, swelling and thixotropic behaviour of myofibrils showed a period in which it was constant or changed slowly, and then began to decrease. The results obtained by polyacrylamide gel electrophoresis indicated that during storage the formation of aggregates occurred in addition to alteration in the actin-myosin interaction. These results were correlated with those obtained from measurements of viscosity, rheological behaviour and swelling of myofibrils in solution.     

Composition, indigenous proteolytic enzymes and coagulating behaviour of ewe milk as affected by somatic cell count

This study was undertaken to assess the effect of somatic cell count in ewe milk on i) composition and hygienic traits; ii) plasmin, cathepsin and elastase activities; iii) leukocyte differential count; iv) renneting parameters. Individual ewe milk samples were grouped according to somatic cell count (SCC) into five classes: SC300 (<300 000 cells/ml), SC500 (from 301 000 to 500 000 cells/ml), SC1000 (from 501 000 to 1 000 000 cells/ml), SC2000 (from 1 001 000 to 2 000 000 cells/ml) and SC>2000 (>2 001 000 cells/ml). Individual milk samples were analysed for pH, chemical composition, microbial features, indigenous proteolytic enzymes, differential leukocyte population, and renneting parameters. Milk yield, lactose, protein, non casein nitrogen, microbial features were affected by SCC level. Plasmin and elastase activities were the highest in samples with more than 1 000 000 cells/ml; plasmin had intermediate values in samples with 300 000 to 1 000 000 cells/ml and the lowest in samples with less than 300 000 cells/ml of milk. Cathepsin D showed significantly lower values in SC300 and SC1000 classes than in SC500, SC2000 and SC>2000 classes. The highest percentages of lymphocyte were found in samples with less than 1 000 000 cells/ml, while the highest levels of polymorphonuclear leukocyte were found in samples with more than 1 000 000 cells/ml of milk. Longer clotting time was found in SC>2000 samples, while reduced clot firmness was observed in SC500 and SC>2000 samples. Results on milk yield and on compositional parameters evidenced an impairment of udder efficiency in ewe milk samples starting from 300 000 cells/ml. Plasmin activity in milk can be considered as a marker of the synthetic and secreting ability of the mammary gland; furthermore plasmin and elastase were consistent with the health status of the udder. Finally cathepsin D played a role in the worsening of renneting properties of ewe milk.     

Kinetics of heat-induced whey protein denaturation and aggregation in skim milks with adjusted whey protein concentration

Microfiltration and ultrafiltration were used to manufacture skim milks with an increased or reduced concentration of whey proteins, while keeping the casein and milk salts concentrations constant. The skim milks were heated on a pilot-scale UHT plant at 80, 90 and 120 degrees C. The heat-induced denaturation and aggregation of beta-lactoglobulin (beta-lg), alpha-lactalbumin (alpha-la) and bovine serum albumin (BSA) were quantified by polyacrylamide gel electrophoresis. Apparent rate constants and reaction orders were calculated for beta-lg, alpha-la and BSA denaturation. Rates of beta-lg, alpha-la and BSA denaturation increased with increasing whey protein concentration. The rate of alpha-la and BSA denaturation was affected to a greater extent than beta-lg by the change in whey protein concentration. After heating at 120 degrees C for 160 s, the concentration of beta-lg and alpha-la associated with the casein micelles increased as the initial concentration of whey proteins increased.     

High pressure treatment of bovine milk: effects on casein micelles and whey proteins

Effects of high pressure (HP) on average casein micelle size and denaturation of alpha-lactalbumin (alpha-la) and beta-lactoglobulin (beta-lg) in raw skim bovine milk were studied over a range of conditions. Micelle size was not influenced by treatment at pressures <200 MPa, but treatment at 250 MPa increased micelle size by approximately 25%, while treatment at > or = 300 MPa irreversibly reduced it to approximately 50% of that in untreated milk. The increase in micelle size after treatment at 250 MPa was greater with increasing treatment time and temperature and milk pH. Treatment times > or = 2 min at 400 MPa resulted in similar levels of micelle disruption, but increasing milk pH to 7.0 partially stabilised micelles against HP-induced disruption. Denaturation of alpha-la did not occur < or = 400 MPa, whereas beta-lg was denatured at pressures >100 MPa. Denaturation of alpha-la and beta-lg increased with increasing pressure, treatment time and temperature and milk pH. The majority of denatured beta-lg was apparently associated with casein micelles. These effects of HP on casein micelles and whey proteins in milk may have significant implications for properties of products made from HP-treated milk.